The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference.
Prostate-specific antigen (PSA; also designated hK3) and human glandular kallikrein 2 (hK2) are two closely related serine proteases highly expressed, predominantly in prostatic tissue. [Wang et al. Invest Urol 1979; 17:159-163, Lilja J Clin Invest 1985; 76:1899-1903, Chapdelaine et al. FEBS Lett 1988; 236:205-208]. The PSA gene is located on the long arm of chromosome 19 and has >84% nucleotide sequence identity with hK2. The two proteins also show extensive similarity in amino-acid sequence (79%) but the expression rates are quite different (hK2 mRNA levels amount to ˜10-20% of PSA mRNA levels) [Schedlich et al. DNA 1987; 6:429-437]. PSA is synthesized as a 261 amino acid preproform from which the 17-21 amino acid signal peptide is cleaved and released in the secretion process. The remaining zymogen form of PSA is activated to an active serine protease by cleavage of 3-7 amino acid propeptide [Lövgren et al. Biochem. Biophys. Res. Comm. 1997; 238; 549-555]. Recently, recombinant hK2 was shown to convert in vitro inactive recombinant proPSA into active mature PSA [Lövgren et al. Biochem Biophys Res Commun 1997; 238:549-555, Takayama et al. J Biol Chem 1997; 272:21582-21588, Kumar et al. Cancer Res 1997; 57:3111-3114]. Therefore, hK2 is likely a physiological activator of proPSA. Enzymatically active PSA is secreted into seminal fluid at high concentrations (0.2-5 mg/mL) [Christensson et al. Eur J Biochem 1990; 194:755-763, Ahlgren et al. 1995 J Androl 16:491-498]. In semen, PSA degrades the seminal vesicle derived gel-forming proteins semenogelin I and II, causing liquefaction of semen and release of progressively motile spermatozoa [Lilja J Clin Invest 1985; 76:1899-1903]. The action of PSA generates hydrolysis of peptide bonds, mainly C-terminal, of certain tyrosine- and glutamine residues in semenogelin I and II [Malm et al. The Prostate 2000; In press]. By contrast, hK2 generates distinctly different cleavage patterns in semenogelin I and II compared to those generated by the action of PSA, though it is presently unclear whether the hK2 action on the gel proteins has physiological significance [Lövgren et al. Eur. J. Biochem. 1999; 262; 781-789].
Enzymatically active PSA has been shown to manifest unique substrate specificity with limited similarity to chymotrypsin-like proteases [Lilja et al. J Biol Chem 1989; 264:1894-1900 Christensson et al. Eur J Biochem 1990; 194:755-763, Malm et al. The Prostate 2000; In press]. The active single-chain form of PSA forms stable covalent complexes with several extracellular protease inhibitors, such as α1-antichymotrypsin (ACT), α2-macroglobulin (AMG), pregnancy-zone protein (PZP), protein C inhibitor (PCI), and α1-antitrypsin (API) [Christensson et al. Eur J Biochem 1990; 194:755-763, Stenman et al. Cancer Res 1991; 51:222-226, España et al. Thromb Res 1991; 64:309-320, Christensson and Lilja Eur J Biochem 1994; 220:45-53, Zhang et al. Prostate 1997; 33:87-96]. In blood, the predominant immunodetectable form of PSA is covalently linked in complex with ACT and only a minor fraction is in a free, noncomplexed form (PSA-F) [Stenman et al. Cancer Res 1991; 51:222-226, Lilja et al. Clin Chem 1991; 37:1618-1625].
LNCaP (lymph node cancer of the prostate) is a human metastatic prostate adenocarcinoma cell line that was isolated in 1977 from a needle aspiration biopsy of a patient with confirmed metastatic prostate cancer. Various forms of free PSA have been found in spent cell culture medium of LNCaP cells. Corey et al. and Väisänen et al. reported LNCaP cells to produce zymogen forms of PSA (proPSA) and a mature intact form of PSA. However, the LNCaP cells do not appear to produce any internally cleaved forms of PSA, by contrast to PSA from seminal fluid which partially occurs as enzymatically inactive forms due to internally cleavage(s) mainly between Lys145 and Lys146 [Christensson et al. Eur J Biochem 1990; 194:755-763]. The zymogen form of PSA has been found also in serum of patients with prostate cancer. Since the zymogen form of PSA is enzymatically inactive, it cannot form complexes with serpins and is likely to remain in a free form in the circulation. There are also other, contradictory reports on the nature of the free PSA form occurring in serum, stating that it is a cleaved, inactive form resulting from internal cleavage(s) or that it represents an unclipped mature but enzymatically inactive form of PSA.
The incidence of prostate cancer has increased during the last decade mainly due to prolonged lifetime and increased screening. This fact underlines the need of improved diagnostic approaches and new treatments. Analysis of PSA in serum is well established in the diagnosis and monitoring of prostate-cancer (PCa) patients [Oesterling J Urol 1991; 145:907-923]. However, raised serum concentrations of PSA are also found in patients with other prostatic diseases, for instance benign hyperplasia of the prostate (BPH) [Hudson et al. J Urol 1989; 142:1011-1017]. The discovery of several different molecular forms of PSA in serum have significantly improved the specificity of diagnosis and monitoring for PCa. Patients with BPH have higher proportions of free-to-total PSA (i.e. PSA-F+PSA-ACT+other quantitatively less important PSA-serpin complexes), or free-to-complexed PSA in serum than patients with PCa. This has resulted in the use of free-to-total PSA (also called percent free PSA) to distinguish between BPH and PCa in men with moderately elevated PSA levels in serum [Stenman et al. Cancer Res 1991; 51:222-226, Christensson et al. J Urol 1993; 150:100-105]. Although this has improved the specificity for PCa, there is still considerable overlap between the two groups of men and therefore a great need for markers, which provide further improved discrimination of men with cancer from normal men and men with benign conditions.
Immunizations of mice with purified PSA have resulted in generation of monoclonal antibodies against PSA and hK2. Many monoclonal antibodies cross react with PSA and hK2 due to the extensive identity in primary structure of the two proteins. However, specific immunoassays that selectively measure free PSA, complexed PSA and hK2 have been developed by us and others. At present, there are no immunoassays available that specifically recognize various candidate forms of free PSA.